Patients with glioblastoma (GBM) have a dismal prognosis. We found that over 90% of patients with GBM over-express both interleukin 13 receptor alpha 2 (IL-13RA2) and EphA2 receptor that are absent in normal brain. More recently, we have demonstrated that the EphA3 receptor is also over-expressed in GBM, and its pattern of expression differs from that of EphA2. IL-13RA2, EphA2, and EphA3 are present in various compartments of GBM tumors. All three receptors are expressed in tumor cells of the core of tumor and in locally-infiltrating tumor cells, while EphA2 is also over-expressed in tumor neovasculature. Further, IL-13RA2, EphA2, and EphA3 are associated with, and play crucial roles in, the pathobiology of glioma stem-like cells (GSC). IL-13RA2 contributes to GSC properties and EphA2 and EphA3 both drive their self-renewal and tumorigenicity. Finally, the EphA3 receptor can be readily detected in GBM-infiltrating cells of monocytic origin, tumor-associated macrophages (TAM). Thus, collectively, IL-13RA2, EphA2, and EphA3 are expressed in principal GBM compartments shown to be involved in tumor progression and/or resistance to therapies. One of the Eph receptor ligands, ephrinA5 (eA5), binds EphA2 and EphA3 and also uniquely the EphB2 receptor; the latter also is expressed specifically on GBM cells. In the current project, we will pursue the novel idea of targeting all four receptors with one pharmaceutical compound. This bi-valent compound will recognize IL- 13RA2, EphA2, EphA3, and EphB2 and deliver a drug to GBM tumors, specifically killing tumor cells and cells of the tumor environment promoting its growth. We have also isolated a small peptide, Pep-1L, which specifically recognizes IL-13RA2, induces receptor's internalization and it is bound by intracranial tumors in mice. These and other properties make the peptide a desirable vehicle to augment access of drugs/labels to tumors. We will continue this exciting line of research through two Specific Aims. In the first Aim, we will produce a potent bi-valent cytotoxin, QUAD-CTX, simultaneously targeting the IL-13RA2, EphA2, EphA3, and EphB2 receptors. We will generate a chemical conjugate with a modified chemotherapeutic which can pass the blood-brain barrier (BBB) on its own, termed WP1244. The drug conjugate will be tested in in vitro models of various compartments of GBM and in vivo. In the second Aim, we will exploit a peptide binding to IL-13RA2 for effective targeted systemic or loco-regional chemotherapy, for monitoring of the disruption of the BBB and responses to treatment. We will exploit internalized peptide Pep-1L, which homes to intracranial tumors, for conjugation to WP1244 (Pep-1L-CTX) and test it in GBM models expressing IL-13RA2 to verify the conjugate's ability to penetrate the BBB and/or blood-brain tumor barrier. We will also generate imaging probes based on the peptide. We expect that this comprehensive assault on GBM will translate into clear-cut durable responses in patients.